The present application is the national stage under 35 U.S.C. 371 of PCT/FR97/02126, filed Nov. 25, 1997.
The invention relates to new polypeptides corresponding to mutant forms of the soluble LAG-3 protein or of its fragments.
The lymphocyte activation gene 3 (LAG-3) expressed in activated T cells and human NK cells encodes a type I membrane protein of 498 amino acids with four extracellular domains of the immunoglobulin superfamily (IgSF) (1). The analysis of its sequence has revealed numerous identities with series of amino acid sequences present at corresponding positions on the CD4 receptor, although the overall sequence homology between these two molecules is barely above a base level (approximately 20% sequence identity). Internal sequence homologies also exist in the LAG-3 molecule, between domains 1 (D1) and 3 (D3) as well as between domains 2 (D2) and 4 (D4), which suggests that LAG-3 has evolved, like CD4, through a gene duplication from a pre-existing structure composed of two IgSFs (1). LAG-3 and CD4 can therefore be considered as xe2x80x9cfirst cousinsxe2x80x9d in the evolution within the IgSF family (2).
The authors of the present invention have shown in previous studies, using a quantitative test of cell adhesion, that the formation of rosettes between COS-7 cells transfected with LAG-3 and B lymphocytes expressing the major histocompatibility complex class II (MHC) molecules is specifically dependent on the interaction between LAG-3 and the MHC class II molecules (2). A direct and specific binding of LAG-3 to various human class II molecules (including various alleles and isotypes) as well as to murine and monkey class II molecules has also been observed with a LAG-3Ig fusion protein (3). This dimeric recombinant globulin LAG-3Ig binds to the monomorphic residue of the MHC class II molecules with a greater affinity (Kd=60 nM at 37xc2x0 C.) than CD4-Ig (4); LAG-3Ig is in fact capable of blocking the class II molecule/CD4 interaction in a test of intracellular adhesion (4).
The role of the LAG-3/class II molecule interaction was studied using monoclonal antibodies specific for LAG-3 (5) and LAG-3Ig molecules (6). This interaction leads to a negative regulation of the activation of T cell clones. The production of a LAG-3/class II molecule interaction is induced by contacts between T cells, probably via a negative signal from the class II molecules in the T cell.
In general, LAG-3 is only expressed after lymphocyte activation both in vivo and in vitro (5) and does not therefore play a role in the response inducing phase, unlike CD4. Moreover, blocking experiments with monoclonal antibodies have shown that LAG-3 does not participate in the phase for recognition by restricted CD4 T cell clones by the class II molecules. The functional role of LAG-3 is therefore substantially different from that of the other ligands for the MHC molecules, CD4 and CD8.
In patent application PCT/FR95/00593, the authors of the present invention have shown that some soluble polypeptide fractions of the LAG-3 protein of SEQ ID NO:11 were also capable of binding to the class II molecules. They have also underlined the potential importance of the amino acids of the region between residue 46 and residue 77, and in particular positions 73, 75, 76 and 77.
The authors of the invention have now sought to characterize precisely the region(s) of LAG-3 specifically involved in the binding to the class II molecules, and, to do this, have synthesized several mutated forms of LAG-3 possessing targeted mutations in the first two domains of the extracytoplasmic region of the mature protein, that is to say the protein free of signal peptide, having the N-terminal sequence L-Q-P-G-A-E (residues 1-6 of SEQ ID NO. 2). The D1 domain extends from amino acid No. 1 (L), amino acid No. 149 (M); the D2 domain extends from amino acid No. 150 (T) to 239 (G). Surprisingly, the authors of the invention have shown that the substitution of a single amino acid was able to induce well-known modifications in the affinity of LAG-3 for the class II molecules, either by substantially increasing the binding of LAG-3 to these proteins, or on the contrary by inhibiting it partially or totally.
One aspect of the present invention is therefore to provide a purified polypeptide corresponding to a mutated form of the soluble LAG-3 protein or of one of its fragments comprising the extracellular domain D1 and D2 consisting:
either in an amino acid substitution at one of the positions selected from the group consisting of:
position 73 where arginine is replaced by glutamic acid (R73E),
position 75 where arginine is replaced by alanine (R75A) or by glutamic acid (R75E),
position 76 where arginine is replaced by glutamic acid (R76E), or a combination of two or three of the preceding substitutions,
or in an amino acid substitution at one of the positions selected from the group consisting of:
position 30 where aspartic acid is replaced by alanine (D30A),
position 56 where histidine is replaced by alanine (H56A),
position 77 where tyrosine is replaced by phenylalanine (Y77F),
position 88 where arginine is replaced by alanine (R88A),
position 103 where arginine is replaced by alanine (R103A),
position 109 where aspartic acid is replaced by glutamic acid (D109E),
position 115 where arginine is replaced by alanine (R115A);
or a deletion of the region between position 54 (P) and position 66 (A).
An other aspect is also the use of these mutants of soluble LAG-3 for the manufacture of medicaments, in particular medicaments which are peptidomimetic for the LAG-3 molecule, that is to say which bind in a similar manner to the MHC class II molecules and which have the same type of immunosuppressive activity as the LAG-3 molecule.
The invention also relates to pharmaceutical compositions comprising, as active ingredient, one of the mutated forms of the LAG-3 protein defined above.
In the text which follows, the polypeptides defined above will be called xe2x80x9cmutants of LAG-3xe2x80x9d.
This definition applies to the polypeptides corresponding to the mutated forms of the whole soluble LAG-3 protein or of fragments of LAG-3 comprising the extracellular domains Di and D2, in particular the fragment composed of the two domains D1 and D2.
The soluble LAG-3 protein corresponds to the sequence between the first N-terminal residue and residue 412 of the mature protein (free of signal peptide).
According to a first embodiment, the invention relates to purified polypeptide mutants of soluble LAG-3 having an affinity for binding to the MHC class II molecules which is greater than the affinity of the wild-type LAG-3 protein. The mutations induced in these peptides are preferably located at the level of the external loop of LAG-3, which is located between residue 46 and residue 77.
More precisely, the invention relates to a purified polypeptide corresponding to a mutated form of the soluble LAG-3 protein or of one of its fragments comprising the two extracellular domains D1 and D2, consisting in an amino acid substitution at one of the positions selected from the group consisting of:
position 73 where arginine is replaced by glutamic acid (R73E),
position 75 where arginine is replaced by alanine (R75A) or by glutamic acid (R75E),
position 76 where arginine is replaced by glutamic acid (R76E), or a combination of two or three of the preceding substitutions.
Preferably, the mutants of LAG-3 result from a combination of two or three substitutions selected from the group consisting of:
(R73E+R75A),
(R73E+R76E),
(R75A+R76E),
(R73E+R75A+R76E)
These different mutants will be called hereinafter xe2x80x9cpositive mutantsxe2x80x9d.
According to another embodiment, the invention provides a purified polypeptide corresponding to a mutated form of the soluble LAG-3 protein or of one of its fragments comprising the extracellular domain D1 and D2, consisting in an amino acid substitution at one of the positions selected from the group consisting of:
position 30 where aspartic acid is replaced by alanine (D30A),
position 56 where histidine is replaced by alanine (H56A),
position 77 where tyrosine is replaced by phenylalanine (Y77F),
position 88 where arginine is replaced by alanine (R88A),
position 103 where arginine is replaced by alanine (R103A),
position 109 where aspartic acid is replaced by glutamic acid (D109E),
position 115 where arginine is replaced by alanine (R115A);
or a deletion of the region between position 54 (P) and position 66 (A).
These mutants will be called hereinafter xe2x80x9cnegative mutantsxe2x80x9d.
The point mutants of LAG-3 according to the present invention may be obtained by site-directed mutagenesis methods.
The site-directed mutagenesis method, which makes it possible to create at will mutations defined both by their nature and by their position is nowadays well known to persons skilled in the art. Its principle is based on a hybridization of a primer oligonucleotide carrying the desired mutation with a denatured DNA template. After formation of a duplex between the mutated DNA strand produced by extension of the primer and the wild-type DNA strand, successive replication cycles under appropriate conditions make it possible to produce DNA mutated on both strands.
It is possible to consider nowadays that site-directed mutagenesis covers a range of protocols and techniques which persons skilled in the art will be able to select or modify depending on their needs. Briefly, site-directed mutagenesis may be carried out according to three types of approach which, in chronological order, are: the single-stranded technology, the double-stranded technology and the PCR technology; each of these three approaches having several variants which are suited to a greater or lesser degree to the specific objectives and constraints of the experimentalist.
Within the framework of the present invention, the site-directed mutagenesis is carried out on the DNA encoding the soluble LAG-3 protein or one of its fragments. The isolated nucleotide sequences encoding the different mutants of LAG-3, as well as their complementary nucleotide sequences, are another aspect of the invention.
The mutant polypeptides of LAG-3, which result from mutations induced on the LAG-3 gene, are obtained by techniques for producing recombinant products after introducing their coding nucleotide sequence into an appropriate cellular host.
In this case, the nucleotide sequence used is placed under the control of signals allowing its expression in the host selected. The cellular host used may be selected from prokaryotic systems such as bacteria, or eukaryotic systems such as for example yeasts, insect cells, CHO (Chinese hamster ovary cells) or any other advantageously commercially available system. A cellular host preferred for the expression of the polypeptides of the invention consists of the fibroblast line COS-7.
The vector should contain a promoter, signals for initiation and termination of translation, as well as appropriate regions for regulation of transcription. It should be capable of being stably maintained in the cell and may optionally possess particular signals specifying the secretion of the translated protein.
These various control signals are selected as a function of the cellular host used. To this effect, the nucleotide sequences encoding the peptides according to the invention may be inserted into vectors replicating autonomously within the host selected, or integrative vectors for the host selected. Such vectors are prepared according to the methods commonly used by persons skilled in the art, and the clones resulting therefrom may be introduced into an appropriate host by standard methods, such as for example electroporation.
The vectors for expressing the polypeptides of the mutants of LAG-3 which are defined above also form part of the present invention.
In the case of the COS-7 cells, the transfection may be carried out using the vector PCDM8, as described in (2).
The invention relates, in addition, to the cells transfected with these expression vectors. These cells may be obtained by the introduction into host cells of a vector for expressing a mutant of LAG-3 as defined above, followed by the culturing of the said cells under conditions allowing the replication and/or expression of the transfected nucleotide sequence encoding a mutant of LAG-3.
When the mutants of LAG-3 are expressed at the surface of the transfected cells, the latter may be used in cell binding and adhesion tests to study the capacity of these mutants to bind to various ligands, in particular membrane antigens of certain cell populations, in particular the MHC class II molecules.
The host cells can also be used in a method for producing a mutant polypeptide of LAG-3, the method itself being included in the present invention, and being characterized in that the transfected cells are cultured under conditions allowing the expression of a mutant recombinant polypeptide of LAG-3, and in that the said recombinant polypeptide is recovered.
The methods of purification used are known to persons skilled in the art. The recombinant polypeptide may be purified from cell lysates and extracts, from the culture medium supernatant, by methods used individually or in combination, such as fractionation, chromatographic methods, immunoaffinity techniques with the aid of specific mono- or polyclonal antibodies, and the like.
An advantageous variant consists in producing a recombinant mutant of LAG-3 fused with a xe2x80x9ccarrierxe2x80x9d protein, for example an immunoglobulin or an immunoglobulin region, to form a chimeric protein. One of the advantages of this system is that it allows stabilization of and a reduction in proteolysis of the recombinant product and simplification of the purification when the fusion partner possesses affinity for a specific ligand.
Fusion proteins consisting of fragments of the extracytoplasmic domain of LAG-3 which are bound to the joining region xe2x80x94CH2xe2x80x94CH3 of the heavy chain of a human immunoglobulin (Ig) have in particular been described in application PCT/FR95/00593.
Among the mutant peptides of LAG-3 according to the invention, the positive mutants, that is to say those having affinity for the MHC class II molecules which is greater than the wild-type molecule, can be advantageously used for the manufacture of pharmaceutical compositions having an immunomodulatory activity.
Also included in the invention are the said pharmaceutical compositions comprising, as active ingredient, a mutant of LAG-3, combined with a pharmaceutically acceptable vehicle. Such compositions offer a new approach for controlling the immune responses involving a cellular interaction between activated T cells and cells expressing the MHC class II molecules. These compositions are, for example, useful for exerting an immunosuppressive effect in the case of pathologies linked to abnormal or exacerbated immunological reactions such as autoimmune diseases or the rejection of organ transplants.
The pharmaceutical compositions of the invention are also useful for preventing or slowing down tumour growth IN VIVO. Natural T cell immunosuppression within human tumours is now a recognized biological reality. Consequently, small molecules involved in the LAG-3/MHC class II molecule interaction offer a new line of action in anticancer immunotherapy on this type of relationship between a tumour and the host organism.
The therapeutic compositions according to the present invention may be formulated according to the usual techniques. The vehicle may be in a variety of forms depending on the route of administration selected: oral, parenteral, sublingual, rectal or nasal.
In the case of compositions for parenteral administration, the vehicle will generally comprise sterile water as well as other optional ingredients promoting solubility or the preservation property of the composition. The parenteral routes of administration may consist in intravenous, intramuscular or subcutaneous injections.
The therapeutic composition may be of the sustained-release type, in particular for long-term treatments, for example the treatment of autoimmune diseases or for controlling organ transplant rejection. The dose to be administered depends on the subject to be treated, in particular the capacity of their immune system to reach the desired level of protection. The precise quantities of active ingredient to be administered may be determined without difficulty by the practitioner who will initiate the treatment.
The therapeutic compositions according to the invention may comprise, in addition to one or more mutants of LAG-3, another active ingredient, optionally bound by a chemical bond to the mutant of LAG-3. There may be mentioned, by way of example, soluble mutants of LAG-3 according to the invention fused with a toxin: for example ricin or diphtheria toxoid, which are capable of binding to MHC class II molecules and of killing the target cells, for example leukaemia or melanoma cells, or which are fused with a radioisotope.
The use of the negative mutant peptides, that is to say those whose affinity for the MHC class II molecules is totally or partially reduced, illustrates another advantageous aspect of the invention.
The properties of these peptides indeed provide information on the molecular interactions between LAG-3 and the MHC class II molecules, which makes them useful for the manufacture of molecules which are agonists or antagonists for the interaction between LAG-3 and the MHC class II molecules.
As will be illustrated below by the examples, various structures of LAG-3 are probably responsible for the oligomerization and the interaction with the MHC class II molecules. Some negative mutants of LAG-3 may allow oligomerization with the wild-type protein (in soluble form or bound to the membrane), and modify the interaction of the resulting complexes with the MHC class II molecules. Such mutants may thus induce reversion of certain functions of LAG-3. Consequently, it appears that the negative mutants of LAG-3 constitute advantageous tools for developing small-sized agonists or antagonists derived from LAG-3 or the MHC class II molecules.
Other characteristics and advantages of the invention are illustrated by the examples in the remainder of the description, as well as by the figures whose legends are indicated below.